Positron emission tomography with 2-[18F]-fluoro-2-deoxy-glucose studies have demonstrated that tumors take up 10-fold more glucose than adjacent normal tissues in humans. Over-expression of HIF-1&#945;, activation of Ras and loss of p53 tumor suppressor function are associated with the development of human cancers and each of these genetic alterations converge on glycolysis by activating the 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatases (PFKFB). The PFKFB enzymes interconvert fructose-6-phosphate (F6P) and fructose-2,6-bisphosphate (F2,6BP) [unreadable] F2,6BP is an allosteric activator of 6-phosphofructo-1-kinase (PFK1), a rate-limiting enzyme and essential control point in the glycolytic pathway. There are four PFKFB isozymes which are encoded by separate genes (PFKFB1-4) and characterized by distinct kinase:phosphatase activity ratios. PFKFB3 and PFKFB4 are of particular interest since these isozymes have been found to be activated in human cancer cell lines and tumors, and to be increased by hypoxic exposure via HIF-1&#945;. We recently discovered a small molecular inhibitor of PFKFB3 and PFKFB4, 3-(3-pyridinyl)-1-(4-pyridinyl)-2- propen-1-one (termed 3PO), that inhibits the growth of xenograft and oncogene transgenic tumors in mice. In preliminary studies, we examined the cellular localization of the PFKFB3 and PFKFB4 enzymes in several transformed cells and were surprised to find that whereas PFKFB4 localized to the cytoplasm (the site of glycolysis), PFKFB3 localized to the nucleus. We over-expressed PFKFB3 in HeLa cells and observed no change in glucose uptake but rather an increase in cell proliferation as well as the expression of several cell cycle regulators including cyclin dependent kinase (Cdk) 1, Cdc25C and Cyclin D3. Cdk1 and Cdk2 are essential mammalian cell cycle regulators that promote cell cycle progression in part by phosphorylating cell cycle suppressor proteins including p27 and Rb. We found that the purified F2,6BP allosterically activated recombinant Cdk1 and Cdk2 activity using p27 or Rb as phosphorylation targets in vitro. We also found that compound 3PO caused a decrease in glycolysis, Cdk substrate phosphorylation and a G2/M arrest in HeLa cells. We thus postulate that 3PO and its derivatives may have a dual mechanism of action that enables their selective growth inhibitory properties: (i) inhibition of nuclear PFKFB3 which suppresses Cdk activity and G2/M cell cycle progression;and (ii) inhibition of cytoplasmic PFKFB4 which suppresses glycolytic flux at PFK1. We propose to test this hypothesis by conducting the following three specific aims: 1. To determine the effects of over-expression of PFKFB3, PFKFB4, combined PFKFB3/4 or Cdk1 on the inhibitory effects of compound 3PO on glycolysis, Cdk activity and growth;2. To determine the effect of increased Cdk activity on the anti-tumor effects of compound 3PO in vivo;3. To determine the effects of cre-mediated single or dual PFKFB3/PFKFB4 genomic deletion on the glucose uptake, Cdk activity and growth of tumors in vivo.